Chapter 5

Formulation, characterization and

in vitro evaluation of sterically

stabilized liposomes of juglone

Journal of Pharmaceutical Sciences (Under Review)

Chapter 5

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ABSTRACT

In the present study, an attempt was made to formulate, optimize and evaluate

sterically stabilized liposomes of juglone in vitro. Initially, preformulation studies

were carried out by infrared spectroscopy using physical mixtures of juglone with the

other excipients intended to be used in the formulation viz., soya lecithin, cholesterol

and mPEG2000-DSPE. Further, solution stability studies of juglone was also carried

out using aqueous buffers of varying pH in the range of 4 to 9.3. Considering its

simplicity, thin film hydration method was chosen for the formulation of sterically

stabilized liposomes of juglone. As a part of formulation optimization, the effect of

cholesterol content as well as mPEG2000-DSPE content on the various

physicochemical properties of the prepared liposomes like particle size, polydispersity

index, zeta potential, entrapment efficiency as well as in vitro release profiles was

evaluated. Further, the cytotoxic potential of juglone (as free and liposome

encapsulated form) against B16F1 melanoma cells in vitro using the standard MTT

assay was also performed.

From the IR spectra (preformulation studies), the presence of excipients did

not seem to have any significant impact on the stability of juglone. Further, an inverse

correlation between the solution stability of juglone and pH of the buffer used was

observed; with juglone being more stable in acidic conditions (acetate buffer pH 4.0).

Based on these studies, acetate buffer pH 4.0 was chosen as the hydration media for

the formulation of liposomes. Formulation optimization studies were carried out

where the size and polydispersity index of the prepared liposomes was found to

increase with the cholesterol as well as mPEG2000-DSPE content. Further, increasing

the cholesterol content resulted in an increase in mean entrapment efficiency values

from 47.86 to 66.61 as the cholesterol was increased from 9:0.5:0.3 to 9:3:0.3 (soya

lecithin:cholesterol:mPEG2000-DSPE). Further increase in the cholesterol content did

not result in improved entrapment efficiencies. In vitro release studies showed an

inverse correlation between mPEG2000-DSPE content and cumulative % drug release.

Based on these studies, formulation with lipid composition of 9:3:0.6 (soya

lecithin:cholesterol:mPEG2000-DSPE) was considered as the optimum formulation for

further studies and had a mean particle size of 137.1 nm and zeta potential value of -

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45.7 mV. MTT assay revealed that liposomal juglone was more toxic in comparison to

free juglone against B16F1 melanoma cells grown in vitro.

In conclusion, the optimized sterically stabilized liposomal formulation of

juglone exhibited acceptable size, zeta potential, polydispersity index, entrapment

efficiency as well as in vitro drug release with improved cytotoxic potential against

melanoma cells.

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5.1. INTRODUCTION

Quinones have been extensively investigated for their potential as anticancer

compounds and such research efforts have yielded numerous clinically important

molecules like doxorubicin, mitoxantrone, siantopin etc. However, many more potent

anticancer compounds are still under investigation (Kim et al., 2006; Babula et al.,

2007).

Juglone is one such naphthoquinone derived from the roots, leaves, nut-hulls,

bark and wood of black walnut (Juglans nigra L.), European walnut (Juglans regia

L.) and butternut (Juglans cinerea L.) belonging to the family Juglandaceae. The

herbal preparations of walnut have a long history of use in Chinese traditional

medicine for the treatment of various diseases including cancer (Duke and Ayensu,

1985; Funt and Martin, 1993). Several earlier studies have demonstrated the potential

of juglone to inhibit the growth of various tumors using in vivo tumor models (Okada

et al., 1967; Bhargava and Westfall, 1968; Sugie et al., 1998; Ji et al., 2009). Besides,

several recent studies have also shown juglone to possess potent cytotoxic properties

against various cancer cells in vitro (Segura-Aguilar et al., 1992; Cenas et al., 2006;

Chen et al., 2009; Ji et al., 2011). Based on these studies, the cytotoxic potential of

juglone has mainly been attributed to the ability of juglone to induce reactive oxygen

species leading to an altered redox homeostasis in the cell and thereby cause apoptotic

as well as necrotic cell death. In addition, juglone is also known to be a potent

inhibitor of Pin1 (which is a unique Peptidyl-prolyl isomerase that, in concert with

proline-directed kinases, phosphatases, and ubiquitin ligases, controls the cell cycle),

which is known to be over-expressed in many cancer types and has been hypothesized

to be a chemotherapeutic drug target (Chao et al., 2001; Lu and Zhou, 2007; Yeh and

Means, 2007; Fila et al., 2008). However, juglone being a naphthoquinone is also

reported to exert some toxic effects to normal tissues inc luding acute irritant contact

dermatitis (Neri et al., 2006).

Despite excellent anticancer potential, the in vivo efficacy of some quinones is

rather dismal (Loadman et al., 2002; Phillips et al., 2004). Among the several possible

explanations attributed to this lack of in vivo efficacy, the most likely contributing

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cause is their poor tumor distribution owing to rapid metabolism and elimination from

the systemic circulation (Schellens et al., 1994). Furthermore, some quinones are also

known to have narrow therapeutic indices with substantial toxicities to normal tissues

(Smith, 1985). These limitations of the anticancer chemotherapies have been

overcome by the use of various drug delivery approaches that provide selective, and

sufficiently high, localization of „„active‟‟ drug at the tumor site (Sreeramoju and

Libutti, 2010), thereby improving the tumoricidal efficacy and reducing the systemic

toxicity of the entrapped drug. Among the various particulate drug carriers, liposomes

have gained most attention. From the biomedical viewpoint, liposomes are

biocompatible / biodegradable, cause very little or no antigenic, pyrogenic, allergic

and toxic reactions; they protect the host from any undesirable effects of the

encapsulated drug, at the same time protecting the entrapped drugs from the

inactivation under physiological conditions; and, last but not least, liposomes are

capable of delivering their content inside many cells (Torchilin, 2008). From the

perspective of a formulation scientist, liposomes are considered to be versatile as they

can encapsulate both hydrophilic and hydrophobic drugs. Also, they are regarded as

very flexible, in a way that their surfaces can be easily modified with a variety of

functional moieties such as polyethylene glycol (PEG) and targeting ligands

(Moghimi and Szebeni, 2003).

In the previous study (described in chapter 3), the cytotoxic potential of

juglone against B16F1 melanoma cells growing in vitro was attributed to

multifactorial mechanisms including the induction of oxidative stress, cell membrane

damage, and a genotoxic effect leading to cell death by both apoptosis and necrosis.

In the subsequent studies (described in chapter 4), the anticancer and radiosensitizing

potential of juglone was demonstrated both in vivo and in vitro against a chemo- and

radioresistant B16F1 melanoma model.

To our knowledge, no previous attempts have been made to improve the

anticancer potential of juglone with subsequent reduction in its toxic effects using

drug delivery platforms. Keeping this in perspective, present study was designed to

formulate and evaluate the prepared sterically stabilized liposomes of juglone in terms

of its size, zeta potential, polydispersity index, entrapment efficiency as well as in

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vitro drug release profiles. Further, this study was also aimed to evaluate the prepared

liposomes for their in vitro cytotoxicity against B16F1 melanoma cells in comparison

to free juglone.

5.2. MATERIALS AND METHODS

5.2.1. Chemicals and reagents

Juglone, Minimum essential medium (MEM), 3-(4,5-Dimethylthiazol-2-yl)-

2,5-diphenyl tetrazolium bromide (MTT), L-glutamine, gentamycin sulfate, soya

phosphatidylcholine (SPC), cholesterol were obtained from Sigma Chemicals Co.,

(St. Louis, Mo, USA). 1, 2-distearoyl-sn-glycero-3-phosphoethanolamine [methoxy

(polyethyleneglycol)-2000] (mPEG2000-DSPE) was a generous gift from Genzyme

Corporation (Cambridge, MA, USA). Fetal bovine serum (FBS) was purchased from

Genetix Biotech Asia, India. Dimethylsulfoxide (DMSO), methanol and chloroform

were obtained from Rankem laboratories (India). Methanol and all the other reagents

used for HPLC analysis were of HPLC grade and procured from Merck, Mumbai,

India.

5.2.2. Excipient profiles

5.2.2.1. Soya phosphotidyl choline

Synonyms: 1,2-Diacyl-sn-glycero-3-phosphocholine; 3-sn-phosphatidylcholine; L-α-

Lecithin; Azolectin (Figure 5.1)

Biological source: Soyabean

Molecular weight: 776 g/mol

Description: yellow to very dark yellow, soft granular powder

Typical lots of pure soybean phosphatidylcholine have fatty acid contents of

approximately 13% C16:0 (palmitic), 4% C18:0 (stearic), 10% C18:1(oleic), 64%

C18:2 (linoleic), and 6% 18:3 (linolenic) with other fatty acids being minor

contributors.

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Melting point: 188 °C

Storage temperature: 2 - 8 °C

Assay: 14 - 23% based on choline basis

Figure 5.1. Chemical structure of soyaphosphatidyl choline

Phosphatidylcholine is the major membrane phospholipid in eukaryotic cells.

In addition to being the major structural component of cellular membranes,

phosphatidylcholine serves as a reservoir for several lipid messengers. It is the source

of the bioactive lipids lysophosphatidylcholine, phosphatidic acid, diacylglycerol,

lysophosphatidylcholine, platelet activating factor, and arachidonic acid (Kent and

Carman, 1999). An understanding of the control and regulation of the several

metabolic pathways involved in the formation of these bioactive lipids is an ongoing

science. Apart from that, it is used as a main component in the preparation of

liposomes, applicable for sustained or site specific delivery.

5.2.2.2. Cholesterol

Synonyms: Cholesterin; cholesterolum; 3β-Hydroxy-5-cholestene; 5-Cholesten-3β-ol

(Figure 5.2)

Molecular formula: C27H46O

Molecular weight: 386.65 g/mol

Description: Cholesterol occurs as white or faintly yellow, almost odorless, pearly

leaflets, needles, powder, or granules. On prolonged exposure to light and air,

cholesterol acquires a yellow to tan color.

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Melting point: 147 - 150 °C

Stability and Storage: Cholesterol is stable and should be stored in a well-closed

container, protected from light (2-8 °C)

Density: 1.052 g/cm3 for anhydrous form

Assay: ≥95% (GC)

Cholesterol is used in cosmetics and topical pharmaceutical formulations at

concentrations of 0.3–5.0% w/w as an emulsifying agent. It imparts water-absorbing

power to an ointment and has emollient activity.

Figure 5.2. Chemical structure of cholesterol

Cholesterol also has a physiological role. It is the major sterol of the higher

animals, and it is found in all body tissues, especially in the brain (~25% of total brain

lipid is cholesterol) and spinal cord. It is also the main constituent of gallstones.

It is also one of the most important lipids used in the formulation of liposomes

where it is known to impart rigidity to the lipidic bilayer aimed at site specific and

sustained drug delivery.

5.2.2.3. Methoxy polyethylene glycol distearoyl ethanolamine (mPEG2000-DSPE)

Chemical name: N-(carbonyl-methoxypolyethyleneglycol-2000)-1,2-distearoyl-sn-

glycero-3-phosphoethanolamine, Sodium Salt (Figure 5.3)

Molecular formula: C142H280N5O56P

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Molecular weight: (n=45) 2788 (calculated as free form)

Description: White solid powder, agglomerates

Boiling point: >300 °C

Melting point: 188 °C

Storage temperature: Store in closed containers at -20 ± 5 °C

Density: 1.067 g/mL at 25 °C (lit.)

Assay: ≥95% (GC)

Figure 5.3. Chemical structure of Methoxy Polyethylene Glycol Distearoyl

Ethanolamine (mPEG2000-DSPE)

It is used as long circulating carrier in the preparation of liposomes

(PEGylated liposomes) for sustained release.

5.2.3. Cancer cell lines

B16F1 melanoma cells was used throughout this study and were routinely

grown in 25 cm2 T-flasks as mentioned earlier (Chapter 3, section 3.2.2).

5.2.4. Pre-formulation studies

These studies are generally performed to choose the best experimental

conditions in terms of maintaining the stability of the encapsulated drug. Therefore,

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pre-formulation studies were carried out to determine the stability of juglone under

various conditions that may be encountered during the formulation studies.

5.2.4.1. Drug-excipient compatibility studies

Fourier transform infrared (FTIR) spectroscopy

Infrared spectra were recorded in the wave number region from 4000 to 400

cm-1 using Shimadzu FTIR 8300 Spectrophotometer (Shimadzu, Tokyo, Japan). The

procedure consisted of dispersing the sample, either drug alone, excipient alone or a

physical mixture of drug and the excipients in the ratio of 1:1 with KBr (200 - 400

mg) and compressing into discs by applying a 5 ton pressure for 5 min in a hydraulic

press. The pellet was then placed in the light path and the spectrum was recorded.

5.2.4.2. Solution stability studies

Preparation of standard stock solutions

A stock solution of juglone in methanol was prepared at a concentration of 1

mg/ml. The resulting solution was stored in brownish vials at 25 °C to protect from

light.

Stability in aqueous solutions of different pH

The stability of juglone in aqueous solutions of different pH (at 25 °C) was

then investigated in order to evaluate the influence of pH on its stability. For these

studies, a sub-stock of 0.1 mg/ml (100 µg/ml) concentration was prepared from the

methanol stock in aqueous buffers of varying pH such as acetate buffer - pH 4.0,

HEPES buffer - pH 5.5, HEPES buffer - pH 6.5, phosphate buffered saline - pH 7.4

and borate buffer -pH 9.3. At different pre-set time intervals (0, 3, 6, 12, 24 and 72 h),

known volume of these samples were withdrawn, diluted with appropriate volume of

mobile phase to get a concentration of 0.01 mg/ml (10 µg/ml) and analyzed using a

HPLC method. The peak area corresponding to 0 h time interval was considered

100% and the drug content remaining after different time intervals was calculated

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based on the respective peak area in relation to the 0 h time interval. The stability of

juglone in methanol at 25 °C was also evaluated using similar methodology.

Stability in acetate buffer at elevated temperatures

The stability of juglone in acetate buffer pH 4.0 was evaluated at elevated

temperatures (60 °C) in comparison to room temperature. For these studies, 2 sets of

sub-stock of 0.1 mg/ml (100 µg/ml) concentration were prepared from the methanol

stock in acetate buffers pH 4.0, with one set being placed at room temperature and the

other placed in a water bath at 60 °C. At different pre-set time intervals (0, 3, 6, 12, 24

and 72 h), known volume of these samples were withdrawn, diluted with appropriate

volume of mobile phase to get a concentration of 0.01 mg/ml (10 µg/ml) and analyzed

using a HPLC method. The amount of drug remaining at various time intervals was

calculated as described in the previous section.

Chromatographic conditions for assay of juglone

The quantitative analyses of juglone content was performed using Waters

Alliance 2695 separations module with 2487 dual λ absorbance detector plus auto

sampler (Waters Corporation, Maple Street, Milford, MA, USA). All

chromatographic experiments were carried out at room temperature using a reverse-

phased Grace Vydac C18 silica column (250 mm × 4.6 mm, 5 μm) and at a detection

wavelength of 254 nm. The mobile phase consisted of methanol and acetic acid (0.1%

v/v) in the ratio of 60:40 and a flow rate of 1 ml/min. All data were analyzed using

EMPOWER II software (provided with the HPLC setup).

5.2.5. Formulation of sterically stabilized liposomes (SSL) of juglone

5.2.5.1. Thin film formation

Various methods have been described in the literature for the preparation of

liposomes among which the method described by Bangham (Bangham and Horne,

1964) is the simplest and most widely used procedure in various research laboratories.

In this method the lipids are casted as stacks of thin film from their organic solution

using flash rotary evaporator under reduced pressure. The thin film of lipids is

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hydrated with aqueous buffer at a temperature above the phase transition temperature

of lipids. The drug to be encapsulated is included either in the aqueous buffer (for

hydrophilic drugs) or in the lipid film (for lipophilic drugs). Thin film method

produces a heterogeneous population of MLV, which can be sonicated or passed

through high pressure homogenizer followed by extrusion through polycarbonate

filters to produce small and more uniform sized population of liposomes.

Briefly, required quantities of juglone, cholesterol, SPC and mPEG2000-DSPE

were weighed, dissolved in chloroform and transferred to a 100 ml round bottom flask

(RBF). The RBF was then connected to a rotary flash evaporator (BUCHI R-215

Rotavapor, Switzerland) equipped with thermostatically controlled water bath at 40 ºC

and rotated at 150 rpm under reduced pressure until a thin lipid film was obtained.

The process was allowed to continue for additional 30 min until all the solvent is

evaporated and a dried lipid film was formed on the walls of the flask.

5.2.5.2. Thin film hydration

The lipid film was then hydrated in acetate buffer (pH 4.0) at the temperature

above the transition temperature (Tc) of the lipid (56 °C for mPEG2000-DSPE) by

rotating the flask at about 200 rpm for about 1 h. The liposome dispersion was

subjected to sizing using high pressure homogenization (HPH) (EmulsiFlex-C3,

Avestin, Canada) for 8 to 10 cycles (flow rate 40 ml/min) at an operating pressure of

about 10,000 - 12,000 psi. The suspension was allowed to stand undisturbed for about

2 h at room temperature to allow liposomes to anneal and stabilize.

5.2.5.3. Separation of free drug from liposome encapsulated drug

Among the different methods used for separation of free drug from the

liposome encapsulated form, centrifugation method is usually the most widely

reported for hydrophobic drugs. In the present study, the un-entrapped juglone was

separated from the liposomal suspensions by initially centrifuging at 10,000 x g for 10

min, after which the supernatant liposomal dispersion was subjected to ultra-

centrifugation (Sorvall WX Ultra Series Centrifuge, Thermo Scientific, USA) at

1,60,000 x g for 1 h to precipitate the liposomes. The supernatant was separated, the

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pellet re-dispersed in the buffer and stored at 4 °C in air tight glass containers until

further testing.

5.2.6. Physicochemical characterization of the SSL juglone

5.2.6.1. Measurement of vesicle size, polydispersity index (PDI) and zeta potential

The mean vesicle size, PDI and zeta potential of the liposomes were measured

using dynamic laser scattering method using Nano ZS®90 (Malvern Instruments, UK).

This technique measures the time dependent fluctuations in the intensity of scattered

light, which occurs due to brownian motion of the particles. Ana lysis of these

intensity fluctuations enables the determination of the diffusion coefficient of the

particles, which are then converted into size distribution. At a constant temperature of

25 °C, the samples were backscattered at an angle of 173° using a 632.8 nm He-Ne

(red) laser. The nano-ZS automatically adapts to the sample by adjusting the intensity

of the laser and the photomultiplier, thus ensuring reproducibility of the experimental

conditions. Liposomal suspension was diluted 100-fold with double-distilled water

and measurement were carried out at 25 °C, assuming a medium viscosity of 1.0200

and medium refractive index of 1.335. The polydispersity index is a measure of

dispersion homogeneity and ranges from 0 to 1. Values close to 0 indicate a

homogeneous dispersion while those closer to 1 indicate high degree of heterogeneity

(Varshosaz et al., 2009). Each sample was measured twice and the data reported as

mean ± SD of two measurements.

The zeta potential of a particle is the overall charge that the particle acquires in

a particular medium. The knowledge of the zeta potential of a liposome preparation

can help to predict the fate of the liposomes in vivo and to assess the stability of

colloidal systems. Measurement of the zeta potential of samples in the Zetasizer Nano

ZS (Malvern instruments, UK) is done using a combination of laser doppler

velocimetry and phase analysis light scattering (PALS), a patented technique called

M-3 PALS to measure the particles‟ electrophoretic mobility. In this technique, a

voltage is applied across a pair of electrodes at either end of a cell containing the

particle dispersion. Charged particles are attracted to the oppositely charged electrode

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and their velocity is measured and expressed in unit field strength as their

electrophoretic mobility. The liposome samples were diluted (1 in 100) with MilliQ

water and the measurements were carried out at 25 °C in duplicates and expressed as

the mean of the two measurements. The dividing line between stable and unstable

suspensions is generally taken at either +30 mV or -30 mV. Particles with zeta

potentials more positive than +30 mV or more negative than -30 mV are normally

considered stable (Alexopoulou et al., 2006).

5.2.6.2. Morphological assessment using transmission electron microscopy

The morphology of the prepared liposomes was studied using transmission

electron microscopy (TEM). Briefly, the liposomal dispersions were diluted ten-fold

with buffer and adsorbed onto 300-mesh, formvar-coated copper grids (E M Sciences,

USA). After allowing the sample to dry on the grid, samples were directly examined

and photographed using a Hitachi-H7650 TEM (Tokyo, Japan) at an accelerating

voltage of 80 kV.

5.2.6.3. Determination of entrapment efficiency

The entrapment efficiency of juglone into the liposomes was estimated using

HPLC method by measuring the drug content in the supernatant as well as in the

liposome pellet. Initially, the liposome dispersion and the supernatant was subjected

to mild detergent treatment (by vortexing with 1 % triton X-100 for 5 min) resulting

in membrane disruption and consequent release of juglone into the medium. This

solution was then centrifuged at 10,000 rpm for 10 min (to eliminate the lipidic

materials) and the supernatant was diluted appropriately with the mobile phase before

subjecting to HPLC analysis as mentioned in the previously (section 5.2.4.2). The

drug entrapment efficiency (EE) then was calculated using the following equation

(Gomez-Hens and Fernandez-Romero, 2006)

ntrapment fficiency % Clip

(Clip Cfree) × 100

where, Clip and Cfree are the concentration of SSL encapsulated and free drug,

respectively.

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5.2.6.4. In vitro drug release kinetics

The in vitro release studies of juglone from SSL was studied using dialysis sac

method as previously described with minor modifications (Saarinen-Savolainen et al.,

1997; Shazly et al., 2008). Briefly, dialysis bags with molecular weight cutoff of

12000 Daltons (Sigma Aldrich Co., USA) were soaked in distilled water at room

temperature overnight (to remove the preservatives) and rinsed thoroughly with

distilled water before use. A known amount of liposome suspension was placed in the

dialysis bag, sealed from both ends and immersed in a beaker containing 50 ml of

acetate buffer. The beaker was placed on a magnetic stirrer and stirring was

maintained at 100 rpm at 37 °C. At pre-set time intervals, 1 ml aliquots of the

dialysate were withdrawn for analysis and immediately replenished with fresh

medium (to maintain sink conditions). The samples were then passed through 0.22

µM filter, the drug content assessed spectrophotometrically at a wavelength of 430

nm and the results presented as % cumulative drug release as a function of time.

5.2.7. In vitro cytotoxicity evaluation

The in vitro cytotoxic potential of juglone as free and SSL encapsulated form

was studied against B16F1 melanoma cells using standard MTT assay. The procedure

consisted of seeding B16F1 melanoma cells in a 96-well microtiter plate at a density

of 5 x 103 cells per well and incubating overnight at 37 °C to allow cell adhesion.

After overnight incubation, the medium was replaced with fresh medium containing

different concentrations of free or SSL encapsulated juglone and incubated for 24 and

48 h. At the end of treatment, the drug containing media was discarded and incubated

further for 4 h with 200 µl of fresh MTT medium to allow the viable cells to reduce

the yellow MTT into dark blue formazan crystals. Finally, the formazan crystals were

dissolved in 200 µl of dimethyl sulphoxide (DMSO) and the absorbance of individual

wells was then measured at 540 nm using a microplate reader (InfiniteM200, TECAN,

Austria). All experiments were done using quadruplicate wells, repeated on two

independent occasions and the data were plotted as % cell viability versus

concentration.

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5.2.8. Statistical analysis

The data obtained from the experiments was analyzed by GraphPAD Prism

Version 3.00 Software (California, USA). For all studies, either student‟s t-test or one

way ANOVA followed by Bonferroni‟s post-hoc test was used to compare the

significance between various treatments. A P value of < 0.05 was considered as

statistically significant.

5.3. RESULTS

5.3.1. Preformulation studies

5.3.1.1. Drug–excipients compatibility studies

In order to prepare a physically and chemically stable formulation, it is

extremely necessary that the drug be compatible with the excipients that are intended

to be used in the formulation.

Therefore, as a part of pre-formulation study of the sterically stabilized

liposomes, a compatibility study of juglone with the other excipients (lipids) was

carried out using FTIR spectroscopy. The main FTIR peaks for pure juglone was

compared with those of its physical mixture and depicted in Table 5.1, Figure 5.4 and

Figure 5.5.

Table 5.1. FTIR wave numbers of pure juglone & physical mixtures with excipients.

Sample No Composition of the sample Major wave numbers (cm-1)

1 Pure juglone

1637.62, 1593.25, 1290.42,

1220.98, 1149.61, 1091.75,

833.28, 744.55, 621.1

2

Physical mixture of juglone with

other excipients like soya lecithin,

Cholesterol and mPEG2000-DSPE

1739.85, 1639.55, 1597.11,

1288.49, 1226.77, 1151.54,

1057.03, 835.21, 742.62, 621.1

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Figure 5.4. FTIR spectra of pure juglone recorded in region from 4000 – 400 cm-1.

Figure 5.5. FTIR spectra of the physical mixture of juglone with other excipients.

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The spectra recorded using the pure juglone from the present study was

equivalent to the juglone spectra mentioned in the literature (Pouchert, 1985). The

absorption bands found in the area of 3000 – 3100 cm-1 corresponds to the aromatic

C-H stretch which was found to be present in both the pure drug as well as the

physical mixture. In the case of pure juglone, the major wave number at 1637.62 cm-1

corresponds to chelated carbonyl group of the p-benzoquinone with the hydroxy

group at position 5 of juglone. In case of the physical mixture this wave number is

slightly shifted to 1639.55. The other major wave number that appears at 1593.25 in

the pure juglone corresponds to the aromatic skeletal vibration or the C=C stretching

of the aromatic carbon atoms. This peak appeared at a wave number of 1597.11 in the

case of the physical mixture. The C-O stretching of the phenolic grouping in pure

juglone appears at 1290.42, 1220.98 and 1149.61 cm-1 which is also present in the

physical mixture but is slightly muffled. Another peak that appears at wave number

1739.85 cm-1 in the FTIR spectra of physical mixture but is missing in pure juglone

spectra corresponds to the aliphatic ester grouping of the soya lecithin. The other

wave numbers at 833.28 cm-1 and 744.55 cm-1 corresponds to para di-substituted C-H

deformation and mono-substituted C-H deformation respectively, which is also found

to be present in the physical mixture.

Though the major wave numbers of the pure juglone can be seen in physical

mixture also, a marginal interaction between the pure juglone and excipients in the

fingerprint region is discernible from the widening of the absorptions between wave

numbers 1500 – 1000 cm-1. However, based on the fact that no major interactions

were seen from this study, juglone was considered to be compatible with the

excipients.

5.3.1.2. Analytical method development for the analysis of juglone using RP-HPLC

RP-HPLC method was developed for the estimation of juglone from in vitro

studies like solution stability, entrapment efficiency etc. Considering the simplicity,

selectivity, sensitivity and wider applicability, a reverse phase HPLC method with UV

detection was developed. For the determination of the entrapment efficiency of the

liposomes, the peak area of sample was extrapolated from a calibration curve

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prepared using known concentrations of juglone in methanol (Figure 5.6 and Figure

5.7)

Figure 5.6. Showing the typical HPLC chromatogram for standard juglone (5 µg/ml)

Figure 5.7. Calibration curve for juglone using the developed RP-HPLC method

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5.3.1.3. Solution stability studies of juglone - Effect of pH

The solution stability of juglone under different pH (ranging from pH 4.0 to

9.3) conditions was thoroughly investigated (Figure 5.8). Juglone solutions under

acidic conditions (acetate buffer) as well as in methanol showed good stability for

juglone where approximately 94 % of the added drug could be recovered even after

72 h incubation, which indicated that juglone has high intrinsic stability under

moderately acidic condition.

Figure 5.8. A) Solution stability of juglone under different pH conditions at room

temperature B) Degradation rate constants for the pH dependent solution stability

studies of juglone at room temperature

On the other hand, juglone solutions maintained under less acidic conditions

(HEPES buffer, pH 5.5) showed recovery values that were significantly lo wer in

comparison to either methanol or acetate buffer, where after 3, 6, 12, 24 and 72 h, the

percent drug remaining was about 96.7 %, 93.2%, 89.5 %, 82.1 % and 66.4 %

respectively. Solutions maintained under even lesser acidic conditions (HEPES

buffer, pH 6.5) exhibited still lower recoveries in the range of 88.8 %, 79.9 %, 65.8

%, 48.6 % and 19.8 % after incubating for 3, 6, 12, 24 and 72 h respectively. When

juglone solutions were maintained either under neutral conditions (PBS, pH 7.4) or

alkaline conditions (Borate buffer, pH 9.3), the recovery was only 79.4 % and 55.1 %

after only 3 h of incubation. No recovery of juglone was possible at 12 h incubation

interval, indicating substantial impact of pH on the stability of juglone. Further, as

can be seen from figure 5.8b, a gradual increase in the degradation rate constants of

juglone was observed as the pH of the solution was increased from 4.0 (degradation

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rate constant of 0.000492) to 6.5 (degradation rate constant of 0.0219) indicating its

lack of stability in solutions of higher pH.

5.3.1.4. Effect of temperature on the solution stability of juglone in acetate buffer

(pH 4.0)

From the previous study, juglone exhibited high degree of solution stability in

acetate buffer (pH 4.0) at room temperature. Attempts were made to evaluate the

stability of juglone in acetate buffer at elevated temperature (60 °C) in comparison to

room temperature (25 °C).

Figure 5.9. A) Solution stability of juglone in acetate buffer at room temperature (25

°C) and elevated temperature (60 °C) B) Degradation rate constants for the

temperature dependent solution stability studies of juglone in acetate buffer pH 4.0

As can be seen in the figure 5.9, juglone solutions maintained in acetate buffer

at room temperature had recoveries of about 93.5 % even after 72 h. In contrast,

juglone solutions maintained at elevated temperatures had recoveries of 99.3 %, 98.2

%, 92.8 %, 83.1 % and 63.9 % after 3, 6, 12, 24 and 72 h respectively. Based on these

results, it is clearly evident that juglone is not stable in acetate buffer at elevated

temperatures for longer time periods. From the data presented in figure 5.9b, a clear

increase in the degradation rate constant from 0.000722 in case of acetate buffer at

room temperature to 0.00638 in the case of acetate buffer at elevated temperature of

60 °C could be seen, indicating the lack of stability of juglone in solutions at elevated

temperatures for extended periods of time.

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5.3.2. Formulation optimization

5.3.2.1. Effect of Cholesterol content on the physicochemical properties of the

prepared liposomes

Initial pilot studies were carried out to determine the optimum molar ratios of

drug (juglone) to lipid and based on these results, a molar ratio of 1:20 (for

juglone:lipid) was found to be optimum (data not shown) and was used throughout

this study. Subsequently, as a part of formulation optimization, several batches of

liposomes with varying amounts of cholesterol were formulated and the effect on

various physicochemical properties including the particle size, polydispersity index,

zeta potential as well as the entrapment efficiency were tested (Table 5.2).

Table 5.2. Composition of the juglone SSL and the effect of cholesterol content on

the physicochemical properties of the prepared liposomes

Formulation

Lipid Composition (molar ratio)

(SPC:Cholesterol:mPEG2000-

DSPE)

Particle size*

(mean ± SD) PDI**

Zeta

potential

(mV)

JL1 9:0.5:0.3 99.9 ± 3.74 0.209 -32.1

JL2 9:1:0.3 104.8 ± 0.42 0.216 -32.0

JL3 9:2:0.3 109.6 ± 1.06 0.225 -32.8

JL4 9:3:0.3 116.9 ± 2.12 0.234 -31.6

JL5 9:4:0.3 122.3 ± 5.79 0.247 -34.0

* particle size in nanometers (nm); ** PDI stands for polydispersity index

It is clearly evident from the table 5.2 that, the particle size of the prepared

liposome increased in a cholesterol concentration-dependent manner. However the

other physicochemical parameters (like polydispersity index and zeta potential) only

showed modest increase.

On the other hand, the percent entrapment efficiency of the prepared

liposomes increased significantly from 47.86 ± 1.98 to 66.61 ± 2.70 when cholesterol

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content was increased from 9:0.5:0.3 to 9:3:0.3 (SPC:Cholesterol:mPEG2000-DSPE)

(Figure 5.10). Increasing the cholesterol concentration further beyond did not cause

any improvement in the entrapment efficiency of juglone into the liposomes (Figure

5.10). Based on these studies, 9:3:0.3 molar ratio of SPC:Cholesterol:mPEG2000-

DSPE was chosen for further optimization of mPEG2000-DSPE concentration.

Figure 5.10. Effect of cholesterol content on entrapment efficiency of juglone into

SSL

5.3.2.2. Effect of mPEG2000-DSPE content on the physicochemical properties of the

prepared liposomes

Further formulation trials were carried out to study the effect of mPEG2000-

DSPE content and to attain at the optimum juglone formulation in terms of particle

size (Figure 5.11), polydispersity index, zeta potential (Figure 5.12) as well as the in

vitro release profiles. The composition of these formulations and the effect on various

formulation parameters is depicted in table 5.3. It can be observed that, increasing the

mPEG2000-DSPE concentration from 9:3:0.3 to 9:3:0.6 (SPC:Cholesterol:mPEG2000-

DSPE) caused an increase in both the particle size as well as the polydispersity index

values, which was similar to what was seen in case cholesterol content effect. Such an

increase in the mPEG2000-DSPE concentration also caused the zeta potential values to

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drop from -31.6 mV further down to -43.1 mV, indicating a more physically stable

formulation.

Table 5.3. Composition of the juglone SSL and the effect of mPEG2000-DSPE content

on the physicochemical properties of the prepared liposomes

Formulation

Lipid composition (molar ratio)

(SPC:Cholesterol:mPEG2000-

DSPE)

Particle size*

(mean ± SD) PDI**

Zeta

potential

(mV)

JL6 9:3:0.3 116.9 ± 2.12 0.234 -31.6

JL7 9:3:0.4 123.4 ± 2.89 0.226 -34.5

JL8 9:3:0.5 129.1 ± 2.33 0.231 -38.2

JL9 9:3:0.6 137.1 ± 2.40 0.243 -43.1

* particle size in nanometers (nm); ** PDI stands for polydispersity index

Figure 5.11. Size distribution of the optimized (JL 9) SSL juglone (peak at 137 nm)

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Figure 5.12. Zeta potential distribution of the optimized (JL 9) SSL juglone (peak at –

45.7 mV)

5.3.2.3. Effect of mPEG2000-DSPE content on in vitro release profile SSL juglone

The effect of increasing the mPEG2000-DSPE concentration on the in vitro

release behavior of the prepared liposomes was also examined (Figure 5.13). From the

data presented in figure 5.13, an inverse relation between the mPEG2000-DSPE content

and the in vitro release rate of juglone (from the prepared liposomes) was visibly

evident. The release pattern was clearly a biphasic one with an initial burst phase

followed by a phase of sustained drug release over an extended period of time.

The formulation that had the least mPEG2000-DSPE content (JL6) released

almost 82 % of the entrapped drug in the first 4 h and then continued to release about

90 % of the entrapped drug in 24 h. Further, the formulation with slightly higher

mPEG2000-DSPE content (JL 7 and JL 8) released about 72 % and 68 % juglone after

4 h and about 85 % and 78 % after 24 h. On the other hand, formulation that had the

highest mPEG2000-DSPE content (JL 9) only released about 61 % in 24 hours. Based

on these studies, juglone liposomes (JL 9) formulated using a lipid molar ratio of

9:3:0.6 (SPC:Cholesterol:mPEG2000-DSPE) was chosen as optimum formulation for

all further experiments.

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Figure 5.13. Effect of mPEG2000-DSPE content on the in vitro release of SSL juglone

5.3.2.4. Morphological evaluation

The structural morphology of the optimized liposomal formulations was

evaluated using transmission electron microscopy (TEM) at a magnification range of

20,000 - 30,000 X.

Figure 5.14. Transmission electron microscopic analysis of SSL juglone showing

spherical shaped liposomes

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Figure 5.14 revealed that the liposomes were of moderate sizes ranging 80 to

200 nm and that most of the liposomes formed appeared spherical or slightly

asymmetrical in shape. Although vesicular structure was discernible, the inner

lamellar could not be unambiguously observed. It was clear from TEM studies that

the size of the optimized liposomal formulation was in the range from 80 nm to 200

nm which concurred well with those measured by dynamic light scattering studies.

5.3.3. In vitro cytotoxicity evaluation

The liposome encapsulated juglone was compared with free juglone for its

cytotoxic effect against melanoma cells grown in vitro (Figure 5.15). As shown in

figure 5.15, a concentration-dependent reduction in the viability of melanoma cells

was observed after treatment with liposomal juglone for 48 h. It was observed that the

cytotoxic effect of juglone against melanoma cells was significantly higher when

formulated as liposomes (IC50 value of 4.1 µM) as compared to the free form (IC50

value of 7.9 µM). Similar results were seen in case of 72 h treatment as well, where

treatment of melanoma cells with free and SSL juglone resulted in IC50 values of 7.1

µM and 3.6 µM respectively.

Figure 5.15. Cytotoxic effect of free and SSL juglone against B16F1 melanoma cells

grown in vitro assessed using MTT assay A) after 48 h treatment and B) after 72 h

treatment

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5.4. DISCUSSION

The present study was designed to develop sterically stabilized liposomal

formulation of juglone aimed to improve its anticancer efficacy and to minimize the

toxicity profiles. Preformulation testing is generally the first step in the rational

development of dosage form of a drug substance. Preformulation may be defined as

“investigation of physical and chemical properties of the drug substance alone and

when combined with excipients”. These studies generally focus on those

physicochemical properties of the compound that could affect drug performance and

development of an efficacious dosage form. The ultimate objective of preformulation

testing is to generate information useful to the formulator to choose the correct form

of the drug substance, evaluate its physical and chemical properties, and generate a

thorough understanding of the material‟s stability under the conditions that will lead

to the development of a stable and effective drug delivery system that can be mass-

produced (Niazi, 2006). Therefore, as a part of preformulation studies, juglone was

evaluated for compatibility with other excipients that were intended to be used in the

sterically stabilized liposomal formulation (mainly the lipids). The FTIR spectra of

the pure juglone and that of the physical mixture (1:1 ratio of juglone with the other

excipients) were more or less the same with most of the standard juglone peaks found

also in the spectra of the physical mixture indicating its compatibility with the

excipients intended to be used in the formulation.

The solution stability studies of juglone were then performed by incubating it

in an array of pH buffers. The results of this study clearly revealed that the solution

stability of juglone is dependent on the pH of the buffer in which it is dissolved (more

stable in the acidic pH and less stable in the alkaline pH). This useful information

suggested that alkaline conditions may be avoided during analysis, formulation,

dosing preparation, and other studies of this anticancer drug candidate. Based on the

knowledge that juglone is a weak acid with a pKa value of around 6.96, high amounts

of juglone would remain in unionized state at acidic pH conditions (acetate buffer pH

4.0), which not only improves the stability to juglone but may also increase the drug

entrapment in the lipid bilayer. Based on these studies, acetate buffer (pH 4.0) was

chosen as the hydration buffer for further studies.

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Since the hydration step had to be performed at elevated temperatures of

around 60 °C, the effect of elevated temperature (60 °C) on the stability of juglone in

acetate buffer was evaluated. Juglone was found to be stable for about 12 h at elevated

temperatures beyond which the drug content reduced. However, bearing in mind that

the maximum duration for which juglone would be exposed to elevated temperature is

not more than 1 h, this study suggested that the preparation procedure may not have

any adverse effect on the stability of juglone.

In the present study, thin film hydration technique was chosen considering its

simplicity as well as the fact that it does not introduce any impurities that can affect

the phase behavior and release properties of liposomes (as may occur in the case of

reverse phase evaporation method or detergent dialysis methods) (Parente and Lentz,

1984; Bhardwaj and Burgess, 2010). Given the fact that lipid composition

significantly influences the size, the stability and the encapsulation efficiency of the

liposome, studies were designed to evaluate the effect of different lipid compositions

on the physicochemical properties of the prepared liposomes. To begin with, the

effect of altering the cholesterol content on the physical properties of liposomes was

evaluated. Not surprisingly, an increase in the particle size as well as the

polydispersity index of prepared liposomes was observed as the molar ratio of

cholesterol increased from 9:0.5:0.3 to 9:4:0.3 (SPC:Cholesterol:mPEG2000-DSPE).

However, there was not much change in the zeta potential values between various

formulations. In contrast, increasing the cholesterol content caused significant

increase in the entrapment of juglone into the prepared liposomes up to a

concentration of 9:3:0.3 beyond which no further improvement in the entrapment

efficiency was observed. The observed enhancement in the entrapment e fficiency of

the liposomes up to 9:3:0.3 (SPC:Cholesterol:mPEG2000-DSPE) may be attributed to a

combination of cholesterol- induced increase in hydrophobicity, rigidity and size of

the prepared liposomes (Chan et al., 2004). Moreover, cholesterol content beyond a

certain limit is known to interfere with the closely packed assembly of lipids in the

vesicles, thereby leading to increased membrane fluidity (Kulkarni et al., 1995;

Ramana et al., 2010), which may ultimately result in reduced encapsulation of

hydrophobic molecules like juglone as seen from the present study. Subsequently,

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studies were carried out to determine the effect of altering the mPEG2000-DSPE

concentrations on the physicochemical properties of the liposomes. As expected,

increasing the mPEG2000-DSPE concentration resulted in further increase in the

particle size, polydispersity index values of the prepared liposomes. It is well

documented that incorporation of lipids with high phase transition temperature like

mPEG-DSPE increases the rigidity of the lipid bilayer and reduces the in vitro release

profiles of the entrapped drug, which was observed in the case of sterically stabilized

liposomes of juglone as well.

Liposomes are known to interact with the target cells in many different ways

and thereby altering the uptake patterns of the encapsulated drug. Among the many

factors that affect the nature of such liposome–cell interactions, liposome-related

factors including the composition, size and charge, the presence of targeting

molecules on the liposome surface etc. are known to play a significant part, which

may be critical in determining drug bioavailability to cells and the magnitude of their

cytotoxic effects (Kamps, 2010). In most cases, liposome encapsulated drugs exhibit

similar or less cytotoxic activity compared to free drug, depending on the phase-

transition temperature of phospholipids used in the formulation (Horowitz et al.,

1992; Drummond et al., 2008). Surprisingly, from the present study, the cytotoxic

effect of the liposome encapsulated juglone was higher than the free juglone. The

enhanced chemical stability of juglone in aqueous solutions when administered as

liposome encapsulated form may have contributed to such an increased cytotoxicity

against melanoma cells. In the earlier studies, it was observed that the solution

stability of juglone is pH dependent where at lower acidic pH (around 4) it was found

to be stable for at least 72 h as compared to neutral or alkaline pH conditions where it

was found to rapidly degrade (less than 4 h). This lack of solution stability of

quinones such as juglone has in the past been reported by other groups as well

(Hadjmohammadi and Kamel, 2006; Ossowski et al., 2008). Considering the fact that

the media (MEM) used to grow the cells has a pH of around 7.4, it may be possible

that juglone in the free form has degraded rapidly as opposed to liposomal juglone

where the drug was released slowly over extended period of time, resulting in cells

being exposed to lower doses of juglone for long durations and thereby causing an

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increase in the cytotoxicity of juglone. A similar enhancement in the cytotoxic effect

of liposome encapsulated form has earlier been demonstrated in the case of topotecan

where an unstable lactone ring is known to hydrolyze rapidly to inactive carboxylate

forms (intact lactone ring is an important structural requirement for anticancer

activity) (Jaxel et al., 1989; Giovanella et al., 1991; Liu et al., 2002).

In conclusion, the sterically stabilized liposomes of juglone formulated and

optimized in the present study exhibited acceptable size, zeta potential, polydispersity

index, entrapment efficiency as well as in vitro drug release. Interestingly, the SSL

juglone exhibited higher toxicities against B16F1 melanoma cells in comparison to

free juglone which may be attributed to the improved solution stability of juglone

when formulated as liposomes. Further in vivo studies on the pharmacokinetic,

biodistribution, pharmacodynamic and toxicity profiles of free and SSL juglone are

warranted.

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